The question of which pathways regulate the differentiation of the stratified epidermis has been central to epithelial biology. Transcription is one of the most important regulatory mechanisms controlling the stepwise program of epidermal differentiation. Epidermis has been used as an excellent model for studying the process of cellular differentiation because the cells form a stratified structure during development, and each stratum is easily identified by morphology and expression of specific markers. Our research effort have focused in characterizing the regulation and function of Dlx3 homeobox transcription factor, a member of the murine Dlx family, with essential roles in epidermal, osteogenic and placental development. Transgenic temporal and spatial mis-expression of Dlx3 in the pre-differentiated basal layer caused an abnormal skin phenotype, characterized by cessation of proliferation and premature differentiation of the basal cells judged by the upregulation of expression of late differentiation markers such as loricrin and filaggrin. We are assessing the role of Dlx3 in modulating the cell cycle during the epidermal differentiation process using cultured keratinocytes and mouse models with inducible-ectopic expression of Dlx3. We are also continuing with the characterization of a novel Ca++-binding protein identified in the laboratory.The novel Ca++-binding protein, Scarf (skin calmodulin-related factor) belongs to the calmodulin-like protein family and is specifically expressed in the differentiated layers of the epidermis. Scarf contains four conserved EF-hand motifs. The functionality of Scarf EF-hand domains was assayed with a radioactive Ca++-binding method, determining that the Scarf EF-hand motifs are functional Ca++-binding domains. The Ca++ signaling dependent systems, such as keratinocyte differentiation process, must be finely tuned for rapid and effective response to transient variations in Ca++ concentration. A central role in the transduction of Ca++ signals is played by members of the Ca++-binding proteins. The functionality of the EF-hand motifs may contribute to specificity in the interaction with the target molecules. Thus, it is through the binding of Ca++ by the EF-motifs that these proteins are able to bind or liberate its target interacting proteins and in this way modulate their function. Furthermore, induction of expression was observed upon in vitro differentiation by Ca++ of primary cultured keratinocytes. The functionality of Scarf EF-hand domains was assayed with a radioactive Ca++-binding method, determining that the Scarf EF-hand motifs are functional Ca++-binding domains. Ca++-binding proteins and CaM exert their role through specific interaction with their target proteins. To determine the roles of Scarf during stratification, we set out to identify the target proteins by affinity chromatography and subsequent analysis by mass spectrometry. Several binding factors including isoforms of the phospho-protein binding adaptor 14-3-3, annexins, calreticulin, ERp72, and nucleolin were identified, and their interactions with Scarf were corroborated by co-immunoprecipitation and co-localization analysis. In order to further understand the functions of Scarf and its targets in the epidermis in vivo, we altered the epidermal Ca++ gradient by acute barrier disruption. The change in expression levels of Scarf and its target proteins were determined by immunohistochemistry and western blot analysis. The expression of Scarf, annexins, calreticulin and ERp72 are upregulated by Ca++ gradient disruption whereas the expression of 14-3-3s and nucleolin was reduced. Since annexins, calreticulin and ERp72 have been implicated in Ca++-induced cellular trafficking including the secretion of lamellar bodies and Ca++ homeostasis, we propose that the interaction of Scarf with these proteins might be crucial in the process of barrier restoration. On the other hand, down-regulation of 14-3-3s and nucleolin potentially is involved in the process of keratinocyte differentiation and growth inhibition. We also approached the study on the functional role of Scarf in barrier restoration during wound healing process. We found increased expression and nuclear presence of Scarf in epidermis of the wound edge 3 and 7 days post wounding, entailing Scarfs role in barrier restoration. We propose that Scarf plays a critical role as a Ca++ sensor, regulating the function of its target proteins in a Ca++-dependent manner during epidermal stratification. We are utilizing a mouse model for the targeted deletion of Scarf during embryonic development to understand the in vivo role of Scarf on wound healing and barrier formation.